Cytoplasmic dynein is a multisubunit enzyme complex that transforms chemical energy into motion along microtubules. It plays a wide range of roles in traffic between the endoplasmic reticulum and the Golgi complex and in the transport of membranous organelles. Such transport is essential for chromosome alignment and segregation, for generating and maintaining organelle structure and position, and for facilitating the transfer of material between compartments. The mechanism for assembly into a functional motor is not understood. Moreover there are no high resolution structures for any subunits of dynein. A 10 kDa subunit, here referred to as DLC, is highly conserved among all known dyneins, with 94% sequence identity between Drosophila and human, and is also a component of myosin V. The high conservation suggests a common function of this particular protein in motor complexes of various organisms. The 10 kDa subunit is found to be essential in several cellular processes: embryonic development and oogenesis in Drosophila, nuclear migration in fungus, formation and maintenance of the teguments in the blood fluke, transport of neuronal nitric oxide synthase across axons, and transcriptional regulation during viral infection. The mechanism for these various functions is not known. The purpose of this project is to determine how the structural characteristics of DLC contribute to its multiple regulatory roles. Covalent cross-linking experiments indicate that this polypeptide exists as a dimeric structure within the dynein complex and associates with two other dynein components. The experiments proposed here will focus on using nuclear magnetic resonance spectroscopy to identify the dimer interface, the high resolution structure and dynamics of the dimer, and potential sites of interactions with protein-within the dynein complex and with cargo. Future research is likely to focus on the role of dimerization on the assembly of the complex in vivo, characterization of other subunits of dynein, and their interactions with DLC. The high resolution structure of DLC will not only give clues to its specific and various functions, but more importantly may provide an avenue into structural characterization of the dynein system.
